The effect of possible inhibitors of fluid-phase pinocytosis on LDL uptake and net cholesterol accumulation in wild-type M-CSF-differentiated macrophages.

<p>Wild-type bone marrow-derived macrophages were pretreated 1 h with drug vehicle or the indicated drug. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 6 h. For ¹²⁵I-LDL uptake, macrophages then were incubated with 200 µg/ml ¹²⁵I-LDL and inhibitor for 6 h. All incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor (except the experiment that compares macrophages treated with LDL and dynamin peptide inhibitor with LDL and control peptide) after basal cholesterol values were subtracted from each. The percent inhibition of ¹²⁵I-LDL uptake compares macrophages treated with ¹²⁵I-LDL alone and ¹²⁵I-LDL with inhibitor. Control values for macrophages incubated with LDL alone or ¹²⁵I-LDL alone are indicated in parentheses. For cholesterol accumulation, control values are expressed as nmol net cholesterol accumulation/mg cell protein. For ¹²⁵I-LDL uptake, control values are expressed as µg ¹²⁵I-LDL uptake/mg cell protein. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments except bafilomycin A1-treated macrophages was 15–19% and 82–86%, respectively. Cell-associated and degraded ¹²⁵I-LDL for bafilomycin A1-treated macrophages was 91% and 9%, respectively. * = <i>p</i><0.05. ** = <i>p</i><0.01. *** = <i>p</i><0.001. ND = not determined. “+” indicates a decrease in the number of macropinosomes and “−” indicates no effect on macropinosomes. “−” in percent inhibition columns indicates stimulation rather than inhibition.</p

Macrophage uptake of ¹²⁵I-LDL is non-saturable.

<p>A. LDLR−/− macrophages were incubated with 1 mg/ml of LDL for 0–24 h and cholesterol accumulation was then assessed. B. LDLR−/− macrophages were incubated 24 h with increasing concentrations of ¹²⁵I-LDL, and then ¹²⁵I-LDL uptake was assessed. Uptake values represent the sum of cell-associated and degraded ¹²⁵I-LDL. The range of cell-associated and degraded ¹²⁵I-LDL was 7–13% and 87–93%, respectively.</p

LDL-derived cholesterol accumulation occurs independently of class I PI3K isoforms.

<p>Cholesterol accumulation after 24 h-incubation without or with 1 mg/ml LDL was assessed in M-CSF differentiated macrophages cultured from wild-type, PI3Kγ-KI, PI3Kβ-KI, and PI3Kδ-KI mice.</p

The effect of PI3K inhibitors and cytochalasin D on LDL uptake and net cholesterol accumulation in LDLR −/− macrophages.

<p>LDLR−/− bone marrow-derived macrophages were pretreated 1 h with either drug vehicle, 50 µM LY294002, 100 nm wortmannin, or 4 µg/ml cytochalasin D. For cholesterol accumulation, macrophages then were incubated with 1 mg/ml LDL and inhibitor for 24 h. For ¹²⁵I-LDL uptake, macrophages then were incubated 5 h with 200 µg/ml ¹²⁵I-LDL and inhibitor. All incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor after basal cholesterol values were subtracted from each. The percent inhibition of ¹²⁵I-LDL uptake compares macrophages treated with ¹²⁵I-LDL alone and ¹²⁵I-LDL with inhibitor. ** = <i>p</i><0.01. *** = <i>p</i><0.001. All inhibitors showed almost complete inhibition of macropinosome formation as assessed by phase-microscopy. For LY294002 and wortmannin experiments, control macrophages incubated with LDL or ¹²⁵I-LDL alone showed net cholesterol accumulation and ¹²⁵I-LDL uptake values of 226±8 nmole/mg cell protein and 2.7±0.1 µg/mg cell protein, respectively. For cytochalasin D experiments, control macrophages incubated with LDL or ¹²⁵I-LDL alone showed net cholesterol accumulation and ¹²⁵I-LDL uptake values of 230±2 nmole/mg cell protein and 3.4±0.1 µg/mg cell protein, respectively. The range of cell-associated and degraded ¹²⁵I-LDL for all treatments was 19–33% and 67–81%, respectively.</p

Macropinosome formation is M-CSF dependent.

<p>A and B. Wild-type macrophages were differentiated with M-CSF for 7 days and visualized by phase-contrast microscopy following the treatments described below. Macrophages differentiated with M-CSF were pretreated 30 min with DMSO drug vehicle (A), or 5 µM of cFMS (i.e., M-CSF receptor) tyrosine kinase inhibitor, GW2580 (B). Pretreatment was carried out without either serum or M-CSF. Withdrawal of M-CSF caused disappearance of the macrophage vacuoles. Subsequently, these macrophage cultures were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) without (A) or with GW2580 (B). Macrophages treated with M-CSF without GW2580 showed numerous vacuoles shown to be macropinosomes in Video S1. In contrast, there was complete inhibition of macropinosome formation when macrophage cultures were treated with GW2580 (also see Video S2). Scale bar in B = 75 µm and also applies to A. (C) Wild-type macrophages were incubated 24 h with 1 mg/ml LDL without or with 5 µM GW2580, and then cholesterol accumulation was assessed. Macrophages incubated without LDL had 111±3 nmol cholesterol/mg protein. ** = <i>p</i><0.01.</p

Wild-type and LDL−/− macrophages incubated with LDL accumulate similar levels of cholesterol.

<p>Wild-type and LDLR−/− macrophages were incubated with 1 mg/ml LDL for 24 h and then total cholesterol accumulation was assessed. The baseline cholesterol levels for wild-type and LDLR−/− macrophages were 107±10 nmol cholesterol/mg protein and 122±9 nmol cholesterol/mg protein, respectively.</p

Fluid-phase pinocytosis mediates LDL uptake.

<p>(A) Wild-type macrophages were incubated 6 h with either 25 µg/ml ¹²⁵I-LDL alone, or 25 µg/ml ¹²⁵I-LDL and 500 µg/ml unlabeled LDL. CD36 KO macrophages (ΔCD36) and SRA KO macrophages (ΔSRA) macrophages were incubated 6 h with 25 µg/ml ¹²⁵I-LDL alone. (B) Wild-type macrophages were incubated 6 h with either 25 µg/ml ¹²⁵I-AcLDL alone, or 25 µg/ml ¹²⁵I-AcLDL and 500 µg/ml unlabeled AcLDL. CD36 KO macrophages (ΔCD36) and SRA KO macrophages (ΔSRA) macrophages were incubated 6 h with 25 µg/ml ¹²⁵I-AcLDL alone. Incubations were performed in serum-free medium containing 50 ng/ml M-CSF. Uptake values represent the sum of cell-associated and degraded ¹²⁵I-labeled lipoprotein. The range of cell-associated and degraded ¹²⁵I-LDL was 17–21% and 79–83%, respectively. The range of cell-associated and degraded ¹²⁵I-AcLDL was 16–18% and 82–84%, respectively. ¹²⁵I-LDL uptake was not competed with excess unlabeled LDL consistent with fluid-phase pinocytosis mediating uptake. Statistical tests compare each treatment group with wild-type macrophages incubated with 25 µg/ml ¹²⁵I-AcLDL. * = <i>p</i><0.05. ** = <i>p</i><0.01. *** = <i>p</i><0.001. There was no statistical difference between macrophage groups incubated with ¹²⁵I-LDL.</p

The effect of PI3K inhibitors and cytochalasin D on net cholesterol accumulation in wild-type macrophages.

<p>Wild-type bone marrow-derived macrophages were pretreated 1 h with either drug vehicle, 50 µM LY294002, 100 nm wortmannin, or 4 µg/ml cytochalasin D. Macrophages then were incubated with 1 mg/ml LDL and inhibitor for 6 h. Incubations were performed in serum-free medium containing 50 ng/ml M-CSF. The percent inhibition of net cholesterol accumulation compares macrophages treated with LDL alone and LDL with inhibitor after basal cholesterol values were subtracted from each. ** = <i>p</i><0.01. *** = <i>p</i><0.001. All inhibitors almost completely inhibited macropinosome formation as assessed by phase-microscopy. For the LY294002 experiment, control macrophages incubated with LDL alone showed net cholesterol accumulation of 31±2 nmole/mg cell protein. For the wortmannin and cytochalasin D experiments, control macrophages incubated with LDL alone showed net cholesterol accumulation of 78±2 nmole/mg cell protein.</p

Effect of plasmin on neutrophil activation.

<p>Fluorescence histograms for expression of CD11b/Mac-1 and CD62L/L-selectin on murine neutrophils undergoing stimulation with plasmin, PMA (open histograms, broken lines), or vehicle (open histograms, solid lines) as compared to control IgG (solid histograms).</p

Role of mast cells and lipid mediators for postischemic leukocyte responses.

<p>Cremasteric ruthenium-red-positive mast cells (A; arrows). The number of ruthenium red-positive cells in mice treated with TXA, EACA, aprotinin, cromolyn, MK-886, or BN52021 undergoing I/R (B). Leukocyte firm adherence (C) and transmigration (D) in mast cell-depleted mice as well as in mice treated with cromolyn, BN52021, or MK-886 undergoing I/R (mean±SEM for <i>n</i> = 4–5 per group; #p<0.05, vs. sham; *p<0.05, vs. vehicle).</p